Neuronal apoptosis due to the inadequate supply of neurotrophins is a critical process during neural development and in certain neurodegenerative diseases. The underlying molecular mechanisms of apoptosis are just beginning to be explored. The activation of an immediate early gene, c-Jun, was found to play an important role in sympathetic neuron death after NGF deprivation. Recently, a kinase cascade that leads to c-Jun activation has been discovered. It includes the activation of small GTP binding proteins (Rac1, Cdc42Hs) through GTP- exchange factors (dbl, ost). Cdc42Hs and Rac1 in turn activate a kinase cascade leading sequentially to PAK ( a p21 activated kinase), MEKK (a MAP kinase kinase kinase), MKK4 (a MAP kinase kinase), and JNK, a MAP kinase that specifically phosphorylates and activates c-Jun. JNK, by activating c-Jun, was recently found to be also involved in the apoptosis of NGF-deprived differentiated PC12 cells. This proposal is designed to examine further whether various components of the previously characterized JNK signaling pathway are all involved in the apoptosis of PC12 cells and sympathetic neurons after NGF withdrawal. The proposed research will involve several goals, 1. to determine whether the various known components of the JNK pathway exist and are activated in apoptotic PC12 cells and whether dominant negative proteins and antisense oligodeoxynucleotides that block components of the JNK pathway are able to block apoptosis; 2. to determine whether constitutively active forms of JNK signaling components are able to induce death even in the presence of neurotrophins; and 3. to determine whether TrkA or p75 NGF receptor, or both, are involved in triggering JNK mediated apoptosis. Taken together, these studies will provide insight into the mechanisms that initiate the apoptotic process in neurons.